Chemokinesins and chemotaxins of leukocytes are endogenous chemical self-components which activate and regulate the processes of emigration of leukocytes from the blood stream and their accumulation in tissue in inflammatory processes.
Local injuries in tissue cause an inflammatory reaction. The inflammatory reaction is defined as process which begins with a sublethal tissue injury and ends with complete destruction or complete healing of the tissue.
The inflammatory reaction comprises numerous biological signal processes. Cells and mediators of inflammation belong to the structural equivalents of this cybernetic loop of the information network of the inflammatory process. Like the classical hormones of endocrine glands, the inflammatory mediators are also substances which are present in only minute concentrations as traces in tissues and in blood. For example, it can be shown that a dividing cell can maintain only up to 5,000 of such mediator molecules in a steady state equilibrium in its surrounding medium.
Many different cell types participate in the biological information network. In particular, they include the different tpyes of leukocytes which accumulate at the reaction site of inflammation in the tissue. These leukocyte types can be subdivided into the following groups: Neutrophils, eosinophils, basophils, monocytes (macrophages), lymphocytes and the different types of juvenile forms of these cells.
The complex biosynthesis from a common precursor form of these cells takes place in the bone marrow. After cell formation, they are stored in the bone marrow as juvenile or mature cell forms. From their primary (so-called poietic) storage pools in the bone marrow they are mobilized and recruited as blood leukocytes into the blood stream, when necessary. The emigration of leukocytes from the blood stream and their accumulation at the reaction site of tissue injury is not a passive process. It is an active process and occurs in well-defined emigration sequences of specific leukocyte populations. Therefore, time-dependently, either different homogenous or mixed populations of leukocytes may accumulate at the reaction site of inflammation.
Inflammatory mediators are soluble chemical substances which participate in the activation and regulation of the body's defence-system as carriers of specific information. They are formed at the reaction site of inflammation by humoral or cellular mechanisms. The transmission of their specific information occurs systemically or locally to neighbour or remote target cells. The mode of action of inflammatory mediators is often similar to that of the hormones of known endocrine glands.
The possible existence of inflammatory mediators with defined structure was first shown by Sir Thomas Lewis (1927) "The Vessels of the Human Skin and their Responses", Shaw, London. He showed that the so-called "triple response" can be mimicked by histamine, a substance of known structure. Today, many different kinds of inflammatory mediators are known. Inflammatory mediators can be simple or complex organic molecules, such as histamine, serotonin, prostaglandins, prostacyclins, thomboxans, leukotriens, etc.
Chemotaxis is a reaction, by which the direction of locomotion of cells or organisms is determined by chemical substances in their environment. This definition shows that chemotaxis is not a specific ability of leukocytes alone, but is a basic property of all living organisms.
Apart from directional locomotion to chemical stimuli (chemotaxis), cells can also be influenced chemically in their motility (chemokinesis). This may result in an inhibition or a promotion of their random locomotion. Accordingly, chemokinesis is a reaction by which the motility of cells or organisms migrating randomly randomly is determined by chemical substances in their environment; see W. Rothert, Flora, vol. 88, 1901, p. 371-421.
Positive and negative changes in the migration trace of a cell may result from alterations in cellular speed, probability and frequency of migration or tumbling and direction of locomotion. These changes in the random migration trace are called positive or negative chemokinesis. If all these changes cancel out each other, the substance investigated has an indifferent chemokinetic activity.
Chemotaxis and chemokinesis of leukocytes (leukotaxis and leukokinesis) can be objectively measured and differentiated only in well defined in vitro test systems. In vivo, such a measurement or differentiation of chemotaxis and chemokinesis is possible only with laborious and ingeneous device systems; see I. K. Buckley, Exp. Mol. Pathology, vol. II. p. 402 to 417.
Therefore, for a long time, a main research problem was the development of reproducible in vitro assay systems for chemokinesis and chemotaxis of leukocytes. The assessment in vitro occurs either by direct microscopic observation of single migration steps of single cells concomitantly to a measurement of locomotion speed of cells migrating in a concentration equilibrium or along a concentration gradient of the substance to be investigated. The results obtained have then to be examined as to whether or not they conform to or deviate from basic relations of random walk theory; see S. C. Peterson, P. B. Noble, Biophys. J. vol. 12, (1972), p. 1048 to 1055. When they conform to basic relations of random walk theory, the type of migration is called chemokinesis; If they deviate from the relations, the type of migration of cells is called chemotaxis. Another test system for chemokinesis and chemotaxis has been developed by S. V. Boyden in the form of a filter assay system consisting of a two compartment chamber. Today, many modifications of this assay system exist. In principle, the migration of many cells through the pores of a filter is measured; see J. H. Wissler et al., Eur. J. Immunol. vol. II, p. 90-96.
An alternative test system for the measurement of negative chemokinetic activity of substances uses the inhibition of cell emigration from glass capillaries; see A. R. Rich and M. R. Lewis, Bull. John Hopkins Hosp., vol. 50 (1932), p. 115 to 131. This inhibition is reversible, if the cells after the assay can be shown to be functionally viable. This functional viability has to be demonstrated in a further assay system, i.e. whether or not they are still chemotactically responsive and motile. If the substances investigated are cytotoxic and therefore, the inhibition of migration is positive, then the result of this second assay system is negative: The observed migration inhibition was not caused by reversible chemokinetic activity of the substance under investigation but the motility of the cells was inhibited by irreversible cytotoxic actions and loss in the function of the cells.
V. Menkin, Biochemical Mechanisms in Inflammation, Charles C. Thomas, Springfield, Ill., 1956 has shown that soluble mediators are contributory factors in mechanisms which induce the emigration of blood leukocytes from blood vessels and their accumulation in tissues. He isolated a crystallizable preparation of substances from inflammatory exudates whose nature has not been characterized in detail. However, with this preparation, he could induce an accumulation of leukocytes in tissues. It has been assumed, however, that contamination with bacterial endotoxins and other exogenous substances in the preparations obtained have contributed to the different types of activities displayed. Such exogenous substances, like endotoxins have a strong indirect biological action on blood plasma or blood cells. It is known that on the one hand, endotoxins may activate blood plasma protein systems, such as kinine and complement protein systems. On the other hand, they have a mitogenic effect on mononuclear leukocytes (B-cell mitogens); see Anderson et al. J. Exp. Med. 137 (1973), p. 943 to 953.
As a result of these findings, humoral serum protein preparations with chemotactic and/or chemokinetic activity on leukocytes have been prepared. However, these preparations have been neither molecularly homogeneous nor have they been biologically specific in their action. Nor have they been characterized in detail; see P. C. Wilkinson (ed.) Chemotaxis and Inflammation, Ch. Livingstone, Edinburgh (1974).
Thus, some of these protein preparations also induce a leukocytosis reaction in vivo; see B. Damerau et al., Naunyn-Schmiedberg's Arch. Pharmacol. 302 (1978), p. 45 to 50. Detailed investigations of the mechanisms of formation of humoral chemotaxins for leukocytes derived from serum-proteins have shown their relationship with anaphylatoxin activity which was detected by Friedberger in 1910; see J. A. Jensen, in Ingram, D. G., (ed.): Biological Activities of Complement, Karger, Basle, (1972), p. 132 to 157.
More recently, using modern chromatographical preparation techniques, such biological active humoral trace proteins could be isolated and characterized in molcularly homogenous, crystalline and chemotactically acting form after about 5,000 to 20,000 fold purification; see J. H. Wissler, Eur. J. Immunol., vol. II, (1972), p. 73-96. These protein preparations have neither a leukokinetic activity nor can they mobilize and recruit leukocytes from the bone marrow into blood circulation.
It is these molecular-biological properties, i.e. the distinct cell and action specificity, in which the natural humoral leukotaxin protein preparations prepared and highly purified from contact-activated serum basically differ from less purified natural and, especially, from synthetic low-molecular peptide leukotaxins (formyl-methionyl derivatives etc.). Taxis, kinesis, adhesion, aggregation and, in addition, phagocytosis of leukocytes are concomitantly and indiscriminately induced by such preparations and by the synthetic peptide.
Consequently, it has been postulated that a common receptor in the cell membrane of leukocytes exists which indiscriminately transmits information for taxis, kinesis, adhesion, aggregation and phagocytosis to the cell.
However, further investigation with highly purified, specifically acting natural mediators show that these postulates are not in conformity with reality. This can be directly demonstrated by comparing the activity of the low-molecular synthetic peptide with the highly purified humoral, natural cell and reaction-specific leukotaxin preparations. While the synthetic peptides indiscriminately activate cells to chemotaxis, chemokinesis, adhesion and aggregation, the specific natural humoral leukotaxin protein preparations only induce directional locomotion (chemotaxis) of leukocytes, without influencing their chemokinesis, adhesion, aggregations or phagocytosis responses.
All the mentioned and described preparations for influencing the chemokinesis and chemotaxis of leukocytes are humoral, serum protein-derived chemical substances. In addition, the existence of cellular (cell-secreted) chemotaxins has been shown. Furthermore, a migration-inhibiting activity of cellular origin has been found ("migration-inhibiting factor", MIF). However, the preparations which cause these activities have neither been characterized in detail nor they they been obtained in a form acting in a biologically specific manner. Surveys on the variety of demonstrated biological activities are given in B. R. Bloom and J. R. David (ed.) "In Vitro Methods in Cell-mediated and Tumor Immunities", Academic Press, New York, 1976 and by J. I. Gallin and P. G. Quie (eds.) "Leukocyte Chemotaxis: Methods, Physiology and Clinical Applications", Raven Press, New York 1978.
The literature reveals that cellular chemokinesins have not been investigated or demonstrated so far. The migration inhibition activity of the MiF preparation has not been clearly distinguished from chemotactic activities. As far as they have been investigated, none of these preparations shows biological specificity. For instance, one chemotatic activity is said to be identical to the transfer factor activity; see J. I. Gallin and P. G. Quei, loc. cit. Moreover, it is largely unknown whether or not such cellular activities can be differentiated from serum-derived humoral activities.
It is therefore a primary object of this invention to provide a new class of cellular chemotoxins and chemotropins and leukocytes.
It is another object of this invention to provide a new class of cellular chemokinesins and chemotaxins from leukocytes in highly purified form.
It is another object of this invention to provide a new class of cellular chemokinesins and chemotaxins from leukocytes in physical quantities for practical use.
It is another object of this invention to provide a new class of chemokinesins and chemotaxins from leukocytes, which represent biologically specific, active and naturally acting mediators for the promotion of the motility or the directional migration of leukocytes.
It is another object of this invention to provide a new class of chemokinesins and chemotaxins from leukocytes, which are suitable for specifically influencing inflammatory processes in mammalian (e.g. human) organisms.
It is still another object of this invention to provide a process for producing said obtaining a new class of chemokinesins and chemotaxins from leukocytes in an economical, biotechnically useful and relatively simple manner.
It is still another object of this invention to provide a process for producing and obtaining a new class of chemokinesins and chemotaxins from leukocytes in a highly purified, molecularly homogenous form and in physical quantities for practical use.
It is still another object of this invention to provide a pharmacetical composition for specifically influencing inflammatory processes in the body of mammalians.
These and other objects and advantages of the present invention will be evident from the following description of the invention.